KR20160121295A - Pharmaceutical composition containing mitochondrial division inhibitor for preventing or treating alzheimer's disease - Google Patents

Abstract

The present invention relates to a composite including mitochondrial division inhibitor-1 (Mdivi-1) as an active ingredient and, more particularly, to a composite for preventing, improving, or treating an Alzheimers disease by suppressing reactive oxygen species (ROS) by using the Mdivi-1 which is an inhibitor of dynamin-related protein 1 (DRP1) and preventing neuronal cell death. The composite of the present invention can be used for prevention, improvement, and treatment of the Alzheimers disease by preventing the neuronal cell death by suppressing mitochondrial division and showing an excellent effect regarding improvement of the cognitive ability and memory power.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating Alzheimer's dementia comprising an mitochondrial cleavage inhibitor as an active ingredient. The present invention relates to a pharmaceutical composition for prevention or treatment of Alzheimer's disease,

The present invention relates to a composition for preventing, improving or treating Alzheimer's disease comprising mitochondrial fission inhibitor as an active ingredient.

Alzheimer's disease is the most common cause of dementia due to aging, and Alzheimer's disease is occurring worldwide due to aging. However, the exact treatment has not yet been developed.

Alzheimer's dementia accumulates Aβ (amyloid-β) which causes Alzheimer-induced amyloid precursor protein cleavage, and accumulated Aβ activates mitochondrial division and generates reactive oxygen species (ROS). This activates the action of the BACE1 (beta-amyloid converting enzyme 1) protein which induces Alzheimer's dementia, causes the neuronal cell damage due to activation of BACE1 action, loss of memory and language ability, It causes disorder. In addition to impairment of brain function, it seriously leads to death.

Alzheimer's disease is diagnosed by physical examination, neurological examination, mental status test, daily life function test, laboratory test such as blood test, brain imaging test, and neuropsychological test.

The treatment of Alzheimer's Dementia includes alleviation of symptoms by using acetylcholinesterase inhibitor Aricept, NMDA receptor antagonist, and β-secretase inhibitor, which increase the amount of neurotransmitter that is reduced in the brain of Alzheimer's disease patients There is a way to delay progress. However, treatment with these drugs can not completely prevent progression of the disease and has a problem that side effects are high. In addition, the effect of the drug is maintained for 6 months to 2 years, after which the effect is lost or decreased. When the drug is discontinued, the deterioration of the memory is markedly deteriorated and the memory deterioration that occurred does not return to the previous level.

Currently, the development of therapeutic agents for the prevention of neuronal cell death by inhibiting the division of the mitochondria, particularly of the Alzheimer's disease, is actively under way around the world, but the effect is insufficient or the side effects are severe, I can not. In addition, up to now, many studies have been conducted mainly on in vitro studies and almost no experiments have been conducted in vivo.

Therefore, there is a growing need to develop a new therapeutic agent for preventing Alzheimer's dementia caused by neuronal death.

The present invention provides a composition for preventing, ameliorating, or treating Alzheimer's dementia comprising, as an active ingredient, a mitochondrial cleavage protein inhibitor in order to complement the effect of the therapeutic agent for Alzheimer's dementia caused by neuronal cell death, The purpose.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease, which comprises mitochondrial fission inhibitor as an active ingredient.

In one embodiment of the present invention, the mitochondrial division inhibitor may be Mdivi-1 (mitochondrial division inhibitor-1).

In another embodiment of the present invention, the mitochondrial division inhibitor may be characterized in that it enhances cognitive ability and memory.

In another embodiment of the present invention, the mitochondrial division inhibitor is selected from the group consisting of β-secretase (BACE1, beta-amyloid converting enzyme 1), β-amyloid precursor protein (APP-CTF), and sAPP- form of APP-beta). < / RTI >

In another embodiment of the present invention, the mitochondrial cleavage inhibitor may be characterized by reducing the production of reactive oxygen species (ROS).

In another embodiment of the present invention, the mitochondrial cleavage inhibitor may be characterized by decreasing the amyloid-β plaque number.

The present invention also provides a health functional food composition for preventing or alleviating Alzheimer's dementia comprising an mitochondrial cleavage inhibitor as an active ingredient.

In addition, the invention provides a method of preventing or treating Alzheimer ' s dementia comprising administering to a subject a pharmaceutical composition comprising a pharmaceutically effective amount of a mitochondrial cleavage inhibitor.

In addition, the present invention provides the use of a pharmaceutical composition comprising a mitochondrial cleavage inhibitor for the prevention or treatment of Alzheimer's dementia.

The composition comprising the mitochondrial cleavage inhibitor according to the present invention as an active ingredient inhibits mitochondrial division, thereby inhibiting reactive oxygen species (ROS), reducing Alzheimer's proteins, decreasing Aβ (amyloid-β) It is confirmed that it is effective to improve ability and memory. Accordingly, it is expected that the composition of the present invention can be utilized in various fields for prevention, improvement, and treatment of Alzheimer's dementia.

FIG. 1 shows the results of Morris water maze (MWM) test using Alzheimer's mouse administered with Mdivi-1 (Mitochondrial division inhibitor-1).
FIG. 2 shows passive avoidance test results using Mdivi-1-administered Alzheimer's mice.
FIG. 3 shows Western blotting experiment results using a brain of a mouse administered with Mdivi-1.
Figure 4 shows the results of treatment of primary neuronal hippocampal cell with Mdivi-1 and Aβ (amyloid-β) oligomers.
FIG. 5 shows the effect of inhibiting reactive oxygen species (ROS) by staining with DCF (dichlorofluorescein) dye after treating Mdivi-1 and Aβ oligomers in SH-SY5Y cells.
FIG. 6 shows the effect of inhibiting reactive oxygen species (ROS) by dyeing with HNE antibody in Alzheimer's mouse administered with Mdivi-1.
FIG. 7 shows mitochondrial inhibition by Mitotracker Red dye staining in Alzheimer's mice treated with Mdivi-1.
Figure 8 shows the effect of reducing the number of Aβ plaques in Alzheimer's mice dosed with Mdivi-1.

The present invention provides a composition for preventing, ameliorating, or treating dementia comprising an inhibitor of Drp1 (mitomondrial fission) protein Drp1 (dynamin-related protein 1) as an active ingredient. At this time, the dementia is preferably, but not limited to, Alzheimer's disease.

In one embodiment of the present invention, the Morris water maze experiment and the passive avoidance experiment were performed to examine the cognitive ability and memory improvement effect of Mdivi-1 (Mitochondrial division inhibitor-1) (see Example 1) . Further, Western blotting was performed to confirm the effect of Mdivi-1 on the amount of protein expression, which is the main cause of Alzheimer's dementia (see Example 2). In addition, immunocytochemistry was performed to confirm mitochondrial division inhibition and reduction of reactive oxygen species (ROS) production (see Example 3) of Mdivi-1, mitochondrial division of Mdivi-1 Immunohistochemistry was performed (see Example 4) in order to confirm the effect on inhibition and reduction of Aβ (amyloid-β) plaque number. Lastly, primary neuronal cells (primary neurons) were cultured to examine the length of mitochondria, treated with Mdivi-1 and Aβ oligomers, and stained with mitotracker red dye (see Example 5).

As a result, in the mouse model of Alzheimer's dementia, Mdivi-1 was found to enhance cognitive ability and memory, β-secretase (BACE1, beta-amyloid converting enzyme 1), β- amyloid precursor protein- (ROS) production and reduction of Aβ (amyloid-β) plaques in the presence of the C-terminal fragment or sAPP-β (seceretory form of APP-β) Thus completing the present invention.

Although mitochondrial division is involved in the induction of Parkinson's disease, mitochondrial division further occurs in Alzheimer's dementia than mitochondrial division of Parkinson's disease. In addition, Parkinson's disease and Alzheimer's dementia differ in the way genes, proteins, and mechanisms that cause each disease work separately, and Mdivi-1 acts on both diseases. Since the treatment outcome of both diseases by Mdivi-1 may also be different, a composition comprising Mdivi-1 as an active ingredient may have specificity for the treatment of Alzheimer's dementia separately from Parkinson's disease.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease comprising mitochondrial fission inhibitor as an active ingredient.

The mitochondrial division inhibitor may be Mdivi-1 (Mitochondrial division inhibitor-1), but the present invention is not limited thereto.

In addition, the mitochondrial division inhibitor may be characterized by enhancing cognitive ability and memory, but the present invention is not limited thereto.

In addition, the mitochondrial division inhibitor may be characterized by decreasing the major causative protein of Alzheimer's dementia, preferably beta-secretase (BACE1, beta-amyloid converting enzyme 1), APP-CTF precursor protein-C-terminal fragment, and sAPP-beta (seceretory form of APP-ss), but the present invention is not limited thereto.

In addition, the mitochondrial cleavage inhibitor may be characterized by reducing the production of reactive oxygen species (ROS), and may be one that inhibits neuronal cell death by reducing the production of active oxygen, no.

In addition, the mitochondrial division inhibitor may be characterized by decreasing the Aβ (amyloid-β) plaque number, but the present invention is not limited thereto.

As used herein, the term "prophylactic " means any action that inhibits or delays the onset of Alzheimer's dementia by administration of the composition of the present invention.

As used herein, the term "treatment" refers to any action that alleviates or alleviates symptoms caused by Alzheimer's dementia by administration of the composition of the present invention.

The pharmaceutical composition according to the present invention may contain a pharmaceutically acceptable carrier in addition to the active ingredient. Pharmaceutically acceptable carriers in the pharmaceutical compositions according to the present invention include, but are not limited to, saline, buffered saline, water, glycerol, polyethylene glycol, vegetable oils, isopropyl myristate, ethanol and the like.

When the pharmaceutical composition according to the present invention is formulated, it may be prepared by using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant and the like, which are usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules, troches and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. Various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be added in addition to water and liquid paraffin, which are commonly used simple diluents. .

Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.

The composition according to the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the desired method, and the dose may be determined depending on the condition and weight of the patient, , The type of drug, the route of administration, and the time, but may be suitably selected by those skilled in the art.

The composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level is determined depending on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts.

The composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and body weight of the patient. In general, 0.001 to 150 mg, preferably 0.01 to 100 mg, One to three doses may be administered. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

In another aspect of the present invention, the present invention provides a method for treating Alzheimer ' s dementia comprising administering the above pharmaceutical composition to a subject.

As used herein, the term " individual " refers to a subject in need of treatment for a disease, and more specifically refers to a human or non-human primate, mouse, rat, dog, cat, horse, Of mammals.

Further, the present invention provides a prophylactic or therapeutic use of Alzheimer dementia comprising the above pharmaceutical composition.

In another aspect of the present invention, there is provided a health functional food composition for preventing or ameliorating Alzheimer's disease comprising mitochondrial fission inhibitor as an active ingredient. That is, the health functional food composition according to the present invention can be used simultaneously or separately as a medicine for the treatment of Alzheimer's dementia before or after the onset stage for the prevention or improvement of Alzheimer's dementia.

The term "improvement" as used in the present invention means all actions that at least reduce the degree of symptom associated with the condition being treated.

The health functional food composition according to the present invention reduces the expression of BACE1 protein, decreases active oxygen and inhibits Aβ production. Therefore, the health functional food composition according to the present invention is added to health food supplements such as foods and beverages for the purpose of preventing or improving Alzheimer's dementia .

There is no particular limitation on the kind of the food. Examples of foods to which the active ingredient can be added include dairy products including dairy products, meat, sausage, bread, biscuits, rice cakes, chocolates, snacks, confectionery, pizza, ramen noodles, other noodles, gums, ice cream, , Beverages, alcoholic beverages and vitamin complexes, dairy products, and dairy products, all of which include health functional foods in a conventional sense.

In the health functional food composition according to the present invention, the active ingredient can be directly added to the food or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term ingestion intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range.

The composition for health beverage of the present invention has no particular limitation on the other components other than those containing the active ingredient as an essential ingredient in the indicated ratio and may contain various flavors or natural carbohydrates as an additional ingredient have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above . The ratio of the above-mentioned natural carbohydrate can be appropriately determined by a person skilled in the art.

In addition to the above, the health functional food composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates such as cheese and chocolate, Alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like. These components may be used independently or in combination. The ratios of these additives can also be appropriately selected by those skilled in the art.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

(Mdivi-1 (Mitochondrial division inhibitor-1), a Drin-1 (dynamin-related protein 1) inhibitor, was administered for one month in a mouse model of Alzheimer's dementia in which amyloid beta The loss of capacity and memory was reduced and mitochondrial division due to Drp1 was inhibited by Mdivi-1 through brain tissue staining. Mitochondrial division inhibition was observed in primary neuronal cells through primary neuronal culture, (BACE1) protein expression to produce A [beta]. It was confirmed that the number of Aβ, which is a major cause of Alzheimer 's disease, is toxic.

Example  1. Neurobehavioral experiments: Mdivi -1 to improve cognitive ability and memory

1-1. Morris Underwater Maze Experiment Morris water maze  ( MWM ) test )

After 30 days of administration of the Drp1 protein inhibitor Mdivi-1 (Mitochondrial division inhibitor-1) in a mouse model of Alzheimer's dementia, Morris water maze (MWM ) test.

The Morris water maze (MWM) test is a widely used test for assessing learning and memory ability. It is a space exploration method in which animals must swim using clues to find hidden escapees.

A circular white water tank measuring 100 cm in width and 35 cm in height and a platform of 8 cm in width and 10 cm in height were prepared and the water tank was fixed to the camera in a closed, quiet and dark behavior test room. Next, in order to lock the platform, water of about 20 ° C was filled in the water tank about 1 cm above the platform height, and milk or skim milk was poured to make the water opaque so that the mouse could not recognize the depth of the water. After placing the partition on the east, west, north, south, and west sides of the water tank, signs such as signs were attached to the north, south, east, and west sides so that the mouse could recognize the direction.

The first day did not place the platform in the aquarium and allowed the rats to swim for 60 seconds to adapt to the water. On the second day, the platform was positioned to the position in the program, and it was measured from the east direction to the platform within 120 seconds. If the rats were placed on the platform for 10 seconds, they were considered successful and allowed to rest for 15 minutes. However, if the mouse failed to go up for 120 seconds, the mice were practiced to be artificially on the platform for 10 seconds. I measured 120 seconds again after 15 minutes, and I practiced again for 10 seconds if I could not get on the platform. The third day, the fourth day, and the fifth day were practiced in different directions from the previous day.

After 4 days of training, students were instructed to perform a probe test on the 6th day. Probe test is an experiment to see if the mouse remembers the position of the platform for 60 seconds after removing the platform to see how long the mouse stays and crosses where the platform was.

As shown in FIG. 1A, mice treated with Mdivi-1 for 30 days and mice treated with Mdivi-1 during the 4-day practice period were more likely to have a higher score than the control group, Alzheimer's, Time was shortened. Also, as shown in FIG. 1B, after the platform existing at the target spot was removed on the 5th day, the mice that were administered with the experimental group Mdivi-1 were removed from the control group Alzheimer's mouse It was confirmed that the time to stay at the target point was long. Finally, as a result of examining the swimming speed between the control group Alzheimer's mouse and the experimental group Mdivi-1, there was no difference in the athletic ability between the groups, as shown in Fig. 1C .

Therefore, it was confirmed that cognitive ability and memory were improved through the Morris water maze experiment using Alzheimer mouse administered with Mdivi-1.

1-2. Passive avoidance reaction experiment Passive avoidance test )

As in Example 1-1, Mdivi-1 (Mitochondrial division inhibitor-1), a Drp1 protein inhibitor, was administered to a mouse model of Alzheimer's dementia for 30 days. To confirm the memory ability of the administered Alzheimer's mouse, (Passive avoidance test).

The passive avoidance response experiment is an experiment in which the nocturnal mice instinctively use the likes of dark places to evaluate memory. The mechanism for conducting the passive avoidance response experiment consisted of two chambers - a dark, airtight room and a clear, bright room - with a partition between the two chambers.

On the first day, the mice were placed in a room where the lamp was on and kept bright, and the response was measured for 30 seconds. If a rat enters a dark room, the compartment is closed and the rat is given an electric shock of 0.5 mA for 2 seconds. If the mice did not enter the dark room for 30 seconds, the mice were left free for 2 minutes and when the mice entered the dark room, they were given an electric shock of 0.5 mA for 2 seconds. On the first day, electrical stimulation was given to recognize the fear of electrical stimulation, and short-term memory of electrical stimulation was evaluated after 24 hours. The response was assessed for 5 minutes, and the mice were placed in a bright room and the time it took for the mice to enter the dark room for 5 minutes was measured.

As a result, as shown in FIG. 2, the time taken for the Alzheimer's mouse, which was an experimental group, to enter the dark room, was longer than that of the control group, Alzheimer's mouse. Thus, the memory of the experimental group administered with Mdivi-1 was improved . In the exercise period (Day 1), there was no difference in the time to enter the dark room, and after 24 hours, the control mice had many mice entering the dark room fast, the experimental mice not entering the dark room, He stayed in a bright room and showed a way into a dark room.

Thus, memory impairment was reduced by Mdivi-1, suggesting that the memory of Mdivi-1-treated groups was maintained for a long time.

Example  2. Western blotting  : Mdivi -1 is the major cause of Alzheimer's Dementia

The brain of the Alzheimer's mouse model containing Mdivi-1 was isolated and homogenized, and Western blotting was performed using the extracted brain tissue samples. In this embodiment, BACE1 (beta-amyloid converting enzyme 1 = beta-secretase) and sAPP-beta (seceretory form of APP-beta), beta-amyloid precursor protein-C-terminal fragment) of primary antibody was used and the amount of major protein was expressed by Actin. APP-CTF includes C99 and C83.

As a result, as shown in Fig. 3A, BACE1 and sAPPβ proteins decreased in mice treated with 40 mg of Mdivi-1, and C99, which is a by-product of Aβ, was also decreased by γ-secretase. As a result of graphically showing the protein of BACE1, the BACE1 protein was significantly reduced at 40 mg / kg, as shown in Fig. 3B. Finally, as a result of graphing the sAPP beta protein, the sAPP beta protein significantly decreased at 40 mg / kg as shown in Fig. 3C.

Therefore, it was confirmed that sAPPβ protein, a by-product of APP cleaved by BACE1 (β-secretase) and BACE1 (a major cause of Alzheimer's disease) in the mouse brain of Mdivi-1-administered mice was decreased.

Example  3. Immunocytochemistry  : Mdivi -1 inhibition of mitochondrial division and reduction of active oxygen production

Primary neuronal hippocampal cells and neuronal SH-SY5Y cells were covered on a cover slip, treated with Mdivi-1 at 25 μM or 50 μM for 4 hours, treated with 5 μM Aβ oligomer for 6 hours, ₂ incubator. Then, 100 nM Mitotracker Red and 10 μM H2DCFDA (2 ', 7'-dichlorodihydrofluorescein diacetate, hereinafter referred to as DCF) were treated with primary neuronal hippocampal cells and neuronal cells for 30 minutes, respectively, and stained with CO ₂ incubator at 37 ° C. After washing with PBS, the cells were fixed in 4% paraformaldehyde for 10 min. After washing with PBS, the cells were mounted on VECTASHIELD mounting medium with DAPI on a slide glass. The results were then derived through fluorescence microscopy.

First, the primary neuronal hippocampal cells were treated with Mdivi-1 and stained with Mitotracker Red dye. As shown in FIG. 4A, the length of mitochondria was increased compared to the control without any treatment, whereas in the cells treated with Aβ oligomer, Mitochondrial cleavage occurred more frequently. In the cells treated with Mdivi-1 and Aβ oligomers, mitochondrial cleavage occurred more frequently than control, but mitochondrial cleavage was less affected by Mdivi-1 than with Aβ treatment. In addition, the average length of mitochondria of FIG. 4A was measured as shown in FIG. 4B. The length of mitochondria was increased in cells treated with Mdivi-1 compared with control, and the length was decreased due to mitochondrial cleavage in cells treated with Aβ. In the cells treated with Mdivi-1 and Aβ oligomers, The mitochondrial division was suppressed.

In addition, SH-SY5Y cells were treated with Mdivi-1 and Aβ oligomers and stained with DCF (dichlorofluorescein) dye, which is a reactive oxygen species (ROS) marker. Intensity was compared and as shown in FIG. 5A, And Mdivi-1 did not show a significant difference in expression of DCF, and the expression of DCF was increased in the Aβ treatment group than in the control group, and ROS production was increased. In the group treated with Mdivi-1 and Aβ oligomers, it was confirmed that the production of ROS was inhibited by Mdivi-1. Also, the result of graphically showing the DCF intensity measurement of FIG. 5A was as shown in FIG. 5B. In the Aβ treatment group, fluorescence intensity was increased. In the Mdivi-1 treated group and Mdivi-1 treated group, Mdivi-1 inhibited mitochondrial division and decreased ROS production.

Therefore, it was confirmed that Mdivi-1 is effective for inhibition of cleavage of mitochondria and reduction of ROS production, which are caused by Aβ, which is a toxic substance.

Example  4. Immunohistochemistry  : Mdivi -1 mitochondrial cleavage inhibition, reduced reactive oxygen production, and Aβ plaque  Check the number reduction effect

After completion of the Morris water maze (MWM) test of Example 1-1, the brains of the rats were harvested, fixed in 4% paraformaldehyde, and stored at 4 ° C for 48 hours in 30% sucrose . After the brain was cut to a thickness of 45 μM, the cut brain tissue was stained with Anti-HNE (4-hydroxy-2-nonenal) and Mitotracker Red and the result was obtained by fluorescence microscopy.

As a result of staining brain tissue with anti-HNE, it was confirmed that reactive oxygen species (ROS) was inhibited in Alzheimer's mouse treated with Mdivi-1 as shown in FIG.

As shown in FIG. 6A, reactive oxygen species (ROS) between the control (Alzheimer's and Alzheimer's) and Mdivi-1 (experimental group) was stained with a lipid oxidation marker HNE antibody. As a result, At 40 mg / kg of the population, the oxidative stress level was suppressed and the staining was less. As a result of measuring the intensity of HNE of FIG. 6A, it was confirmed that the intensity of fluorescence was decreased at 40 mg / kg, which is the experimental group, as shown in FIG. 6B. By inhibiting the mitochondrial division, Mdivi-1 was thought to reduce cellular damage by decreasing mitochondrial hyperfractionation and production of reactive oxygen species.

Next, the brain tissue was stained with Mitotracker Red. As shown in FIG. 7, it was confirmed that mitochondrial division was inhibited in Mdivi-1-administered Alzheimer's mouse.

As shown in FIG. 7A, mitochondrial division of mitochondria between mitochondrial red cells and control mice (Alzheimer's and Alzheimer's) and Mdivi-1 (experimental group) was 40 mg / kg Mitochondrial division was inhibited and mitochondria became major, and small division decreased. As shown in FIG. 7B, the length of the mitochondria of FIG. 7A was measured using 3D. As a result, it was confirmed that the length of the mitochondria was increased at 40 mg / kg as compared with the control group. In addition, as a result of confirming the staining concentration of mitochondria, there was no difference in the concentration in each group as shown in Fig. 7C, and it was confirmed that the length of mitochondria did not change due to the change in the amount of mitochondria.

In addition, DAB staining was performed to measure Aβ plaque counts using 6E10 anti-Aβ and 3,3-diaminobenzidine (DAB) staining kit in slice brain tissue. After DAB staining, the number of Aβ plaques stained with a microscope was measured.

As a result, as shown in Fig. 8, it was confirmed that the number of A [beta] plaques was decreased in Alzheimer's mice dosed with Mdivi-1.

As a result of DAB staining using AE 6E10 antibody between the control (Alzheimer's and Alzheimer's) and Mdivi-1 (experimental group) mice, 10 mg / kg of Mdivi-1 At 40 mg / kg, the Aβ plaque was less stained and the number of Aβ plaques was decreased. As shown in FIG. 8B, the number of Aβ plaques was decreased in the hippocampus of FIG. 8A at 10 mg / kg and 40 mg / kg, respectively, as compared with the control group. Finally, the number of Aβ plaques in the cortex of FIG. 8A was measured. As shown in FIG. 8C, the number of Aβ plaques was decreased at 10 mg / kg and 40 mg / kg, respectively, of the Mdivi-1-treated group as compared with the control group.

Thus, it was confirmed that Mdivi-1 is effective in inhibiting mitochondrial division, reducing reactive oxygen production, and decreasing Aβ plaque number.

Example  5. Primary neuronal cell culture

Hippocampus and Cortex tissues were separated from the brain of fetuses after removing the embryos from fetuses of 16-day-old pregnant mice and observing them with a microscope. Each brain tissue (Hippocampus, Cortex) was placed in trypsin for 15 minutes at 37 ° C. Then, FBS (Fetal Bovine Serum) was added to neutralize the action of trypsin, and each brain tissue was mixed with media. Then, the number of all cells (including astrocyte, neuron, and microglia) Respectively. After confirming the number of cells, the cells were spread on a cover slide coated with cells and cultured in a 37 ° C incubator. After 24 hours, the medium was replaced with the same volume of Neurobasal-A medium and cultured for 7-14 days. When neuronal cells grew, Mdivi-1 and Aβ oligomers were treated to identify changes in mitochondrial length, stained with mitotracker red dye, and confirmed by fluorescence microscopy.

As a result, the length of neuronal mitochondria in the Mdivi-1 treated group was increased compared to the control group treated with nothing, and it was confirmed that Mdivi-1 inhibited mitochondrial division in neuron cells (see FIG. 4A).

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (7)

A pharmaceutical composition for preventing or treating Alzheimer's disease, which comprises mitochondrial fission inhibitor as an active ingredient.
The method according to claim 1,
Wherein said mitochondrial division inhibitor is Mdivi-1 (Mitochondrial division inhibitor-1).
The method according to claim 1,
Wherein said mitochondrial cleavage inhibitor enhances cognitive ability and memory.
The method according to claim 1,
The mitochondrial cleavage inhibitor is composed of β-secretase (BACE1, beta-amyloid converting enzyme 1), APP-CTF (β-amyloid precursor protein-C-terminal fragment), and sAPP-β (seceretory form of APP- Lt; RTI ID = 0.0 > 1, < / RTI >
The method according to claim 1,
Wherein the mitochondrial cleavage inhibitor reduces the production of reactive oxygen species (ROS).
The method according to claim 1,
Wherein said mitochondrial cleavage inhibitor reduces amyloid-beta plaques.
A health functional food composition for preventing or ameliorating Alzheimer's disease, which comprises mitochondrial division inhibitor as an active ingredient.

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